The Effect Of Buddlejae Eyedrops On The Apoptosis-related Factors PI3K,Akt And Caspase-9 In Lacrimal Glands Of Experimental Dry Eye Rabbit Model

ObjectiveTo study the effect of Buddlejae eyedrops on the apoptosis-related factors PI3K,Akt and Caspase-9 in lacrimal glands by establishing a model of castrated male New Zealand White RabbitMethodsThirty-six New Zealand White rabbits were randomly divided into A,B,C,D,E and F groups,6 in each group.Including:Group A:Control,Group B:Model group,Group C:lmg/ml Buddlejae Eye Drops,Group D:1.5mg/ml Buddlejae Eye Drops,Group E:3mg/ml Buddlejay Eye Drops,Group F:model with testosterone.C,D and E groups were given Buddlejay Eye Drops of I mg/ml,1.5 mg/ml,and 3 mg/ml respectively,3 times/day;Group F was given 2 mg/kg testosterone propionate injection in rabbit thigh muscles,3 days/time.After 2 months later,SIT were used to test the model of dry eye of castrated rabbits.During the experiment,one experimental animal died in groups D and F.After 4 weeks by drug intervented,the lacrimal glands were submitted for examination.The morphological changes of the lacrimal glands in each group were observed under light microscope.The expression of PI3K,Akt,and Caspase-9 in lacrimal gland tissues was detected by qPCR and Western Blot.The relative expressions of PI3K,Akt and Caspase-9 mRNA and protein in lacrimal gland tissue were detected by qPCR and Western Blot.Results1.HE stain:The lacrimal gland tissue of the model group was unclear,and the acini and lacrimal gland epithelium were of different sizes and disordered arrangement.A large number of acinic atrophy and fusion were observed,and a large number of vacuoles were formed.The lacrimal gland structure was clear in the 3 mg/ml Buddlejay Eye Drops group.The acinus and lacrimal gland epithelium were uniform in size,well-arranged,and normal in morphology,with little vacuolization.Similar to the testosterone group,they were inferior to the control group.2.Immunohistochemistry:(1)The mean gray value of caspase-9 protein in the lacrimal gland tissue was significantly higher in the 3mg/ml Buddlejay Eye Drops group(P<0.01)than that in the model group(P<0.01),and similar to the testosterone group(P>0.05);The average optical density of Caspase-9 protein was lower than that of the model group(P<0.01),similar to the testosterone group and control group(P>0.05);(2)The average gray value of PI3K protein in the lacrimal gland tissue in 3 mg/ml Buddlejay Eye Drops group was lower than that in the model group(P<0.01),and similar to the testosterone group and control group(P>0.05).The average optical density of the PI3K protein was higher than that in model group.P<0.01),but less than the testosterone group and control group(P<0.05).In comparison,the average optical density values among the 1mg/ml and 1.5 mg/ml Buddlejay Eye Drops group,the testosterone group,the control group was similar(P>0.05);(3)The average gray value of the Akt protein in the lacrimal gland after 4 weeks of using 3 mg/ml Buddlejay Eye Drops was lower than that in the model group(P<0.01),compared with the testosterone group and control group was similar(P>0.05);the average optical density of Akt protein was higher than that in model group(P<0.01),similar to the testosterone group and control group(P>0.05).3.qPCR:After using 3mg/ml Buddlejay Eye Drops about 4 weeks,the relative expression of Caspase-9 mRNA in the lacrimal gland was significantly lower than control group(P<0.01);The relative expression of PI3K and Akt mRNA was significantly higher than the model group(P<0.01).4.Western Blot:The relative expression of Caspase-9 protein in the lacrimal gland tissue with the group of 1mg/ml,1.5 mg/ml and 3 mg/ml Buddlejay Eye Drops was lower than model group after 4 weeks(P<0.01);the relative expression of PI3K protein of the 3 mg/ml Buddlejay Eye Drops was higher than the model group(P<0.01);1 mg/ml,1.5 mg/ml and 3 mg/ml Buddlejae eye drops group had a higher expression of Akt protein than the model group(P<0.01).Conclusion1.Buddlejae eye drops can improve the morphological structure of the lacrimal gland in dry eye model of castrated rabbits.2.2.3mg/ml Buddlejay Eye Drops can down-regulate the expression of pro-apoptotic protein Caspase-9 and its mRNA and up-regulate the expression of anti-apoptosis protein of PI3K,Akt and its mRNA.3.1.5mg/ml Buddlejae Eye Drops can up-regulate the expression of pro-apoptosis protein Caspase-9 to a certain extent,but also can up-regulate anti-apoptosis protein PI3K,Ak,but the effect is less obvious to apoptosis-related protein mRNA.